Antiestrogen binding sites in rat liver nuclei

Isolated, intact rat liver nuclei have high-affinity (Kd = 10(-9) M) binding sites that are highly specific for nonsteroidal antiestrogens, especially for compounds of the triphenylethylene series. Nuclear [3H]tamoxifen binding capacity is thermolabile, being most stable at 4 degrees C and rapidly lost at 37 degrees C. More [3H]tamoxifen, however, is specifically bound at incubation temperatures of 25 degrees C and 37 degrees C than at 4 degrees C although prewarming nuclei has no effect, suggesting exchange of [3H]tamoxifen for an unidentified endogeneous ligand. Nuclear antiestrogen binding sites are destroyed by trypsin but not by deoxyribonuclease I or ribonuclease A. The nuclear antiestrogen binding protein is not solubilized by 0.6 M potassium chloride, 2 M sodium chloride, 0.6 M sodium thiocyanate, 3 M urea, 20 mM pyridoxal phosphate, 1% (w/v) digitonin or 2% (w/v) sodium cholate but is extractable by sonication, indicating that it is tightly bound within the nucleus. Rat liver nuclear matrix contains high-affinity (Kd = 10(-9) M) [3H]tamoxifen binding sites present in 5-fold higher concentrations (4.18 pmol/mg DNA) than in intact nuclei (0.78 +/- 0.10 (S.D.) pmol/mg DNA). Low-speed rat liver cytosol (20 000 X g, 30 min) contains high-capacity (955 +/- 405 (S.D.) fmol/mg protein), low-affinity (Kd = 10.9 +/- 4.5 (S.D.) nM) antiestrogen binding sites. In contrast, high-speed cytosol (100 000 X g, 60 min) contains low-capacity (46 +/- 15 (S.D.) fmol/mg protein), high-affinity (Kd = 0.61 +/- 0.20 (S.D.) nM) binding sites. Low-affinity cytosolic sites constitute more than 90% of total liver binding sites, high-affinity cytosolic sites 0.3%-3.2%, and nuclear sites less than 0.5% of total sites.     

Binding of [H]methyltrienolone to androgen receptor in rat liver

The synthetic androgen methyltrienolone is superior to testosterone and androstenedione for the measurement of androgen receptor in tissues where the native ligands are metabolized into inactive derivatives. [³H]Methyltrienolone binds with a high affinity to androgen receptor in cytosol prepared from male rat livers, as the Scatchard analysis revealed that the K_d value was 3.3 · 10⁻⁸ M and the number of binding sites was 35.5 fmol/mg protein. Since methyltrienolone also binds glucocorticoid receptor which exists in rat liver, the apparent binding of androgen receptor is faulty when measured in the presence of glucocorticoid receptor. The binding of methyltrienolone to glucocorticoid receptor can be blocked by the presence of a 100-fold molar excess of unlabeled synthetic glucocorticoid, triamcinolone acetonide, without interfering in its binding to androgen receptor, because triamcinolone does not bind to androgen receptor. Triamcinolone-blocked cytosol exhibited that the K_d value was 2.5 · 10⁻⁸ M and the number of binding sites was 26.3 fmol/mg protein, indicating a reduction to of that in the untreated cytosol. The profile of glycerol gradient centrifiguration indicated that [³H]methyltriemolone-bound receptor migrated in the 8–9 S region in both untreated and triamcinolone-blocked cytosols, but the 8–9 S peak in triamcinolone-blocked cytosol was reduced to about of that of untreated cytosol.     

Regulation of respiration and energy transduction in cytochrome c oxidase isozymes by allosteric effectors

The binding of TNP-ATP (2 or 3-O-(2,4,6-trinitrophenyl)-ATP) to cytochrome c oxidase (COX) from bovine heart and liver and to the two-subunit COX of Paracoccus denitrificans was measured by its change of fluorescence. Three binding sites, two with high (dissociation constant K_d = 0.2 µM) and one with lower affinity (K_d = 0.9 µM), were found at COX from bovine heart and liver, while the Paracoccus enzyme showed only one binding site (K_d = 3.6 µM). The binding of [³⁵S]ATPaS was measured by equilibrium dialysis and revealed seven binding sites at the heart enzyme (K_d = 7.5 µM) and six at the liver enzyme (K_d = 12 µM). The Paracoccus enzyme had only one binding site (K_d = 16 µM). The effect of variable intraliposomal ATP/ADP ratios, but at constant total concentration of [ATP + ADP] = 5 mM, on the H⁺/e^- stoichiometry of reconstituted COX from bovine heart and liver were studied. Above 98% ATP the H⁺/e^- stoichiometry of the heart enzyme decreased to about half of the value measured at 100% ATP. In contrast, the H⁺/e^- stoichiometry of the liver enzyme was not influenced by the ATP/ADP ratio. It is suggested that high intramitochondrial ATP/ADP ratios, corresponding to low cellular work load, will decrease the efficiency of energy transduction and result in elevated thermogenesis for the maintenance of body temperature. (Mol Cell Biochem 174: 131–135, 1997)     

High affinity estrogen binding by rabbit liver

Macromolecular binding components for [³H]estradiol-17β are present to cytosol prepared from rabbit liver. When cytosol from sexually mature male liver was incubated with [³H]estradiol and analyzed for binding on low ionic strength sucrose gradients, two peaks of binding activity were detected. One peak had a sedimentation coefficient of 4–5 S and the other had a sedimentation coefficient of 8–9 S. The two components differed from each other regarding steroid specicity and various physiocochemical parameters. [³H]-estradiol binding to the 4–5 S component was not inhibited by estrogens, 5α-dihydrotestosterone, progesterone or cortisol. Binding to this component did not appera to be saturable and lavel was rapidly stripped from it by cahrcoal. Estradiol bindng to the 8–9 S component was estrogen specific, saturable and of high affinity. The specific binder dissociates on high ionic strength sucrose gradients and sediments as a 4–5 S moiety. The specific binding protein has a K_d of 3.05 · 10⁻¹⁰ M and a dissociation half-time of 33 h and there are 35.2 fmol of binding sites/mg cytosol protein. Estrogen binders are also present in liver cytosol from sexually mature female and sexually immature male rabbits. During prolonged incbuation of [³H]estradiol with mature male liver cytosol at 0–5°C polar metabolites of estradiol are produced.     

Complexation of lithium(I), sodium(I), silver(I) and thallium(I) by 4,7,13,16-tetraoxa-1,10-diazabicyclo[8.5.5]eicosane and 4, 7, 13-trioxa-1,10-diazabicyclo[8.5.5]eicosane in trimethyl phosphate. A potentiometric titration and Li and Na nuclear magnetic resonance study

Complexation of M⁺=Li⁺, Na⁺, Ag⁺ and TI⁺ by the cryptands 4, 7, 13, 18-tetraoxa-l, 10-diazabicyclo[8.5.5]eicosane (C211) and 4,7,13-trioxa-1,10-diazabicyclo[8.5.5]eicosane (C21C₅) to form the cryptates [M.C211]⁺ and [M.C21C₅]⁺ has been studied in trimethyl phosphate by potentiometric titration and ⁷Li and ²³Na NMR spectroscopy. For [M.C211]⁺ the logarithm of the apparent stability constants, log K (dm³ mol^-1)=6.98±0.05, 5.38±0.05, 9.82±0.02 and 3.95±0.02 for M⁺ =Li⁺, Na⁺, Ag⁺ and TI⁺, respectively; and for [M.C21C₅]⁺ log K (dm³ mol^-1)=2.40±0.10, 1.90±0.05, 6.04±0.02 and 2.42±0.10 for M⁺=Li⁺, Na⁺, Ag⁺ and Tl⁺, respectively. The decomplexation kinetic parameters for [Na.C211]⁺ are: k_d (298.2 K)=6.924±0.50 s^-l, ΔH_d≠=62.2±0.9 kJ mol^-1, and ΔS_d≠= -20.3±2.7 J K^-1 mol^-1; and those for [Li.C21C₅]⁺ are: k_d (298.2 K)=23.3±0.4 s^-1, ΔH_d≠ =61.2±1.1 kJ mol^-1, and ΔS_d≠= -13.6±3.6 J K^-1 mol^-1. Metal ion exchange on [Li.C211]⁺ is in the very slow extreme of the NMR timescale up to 390 K and k_d « 4 s^-1 at 298.2 K, while in contrast exchange on [Na.C21C₅]⁺ is in the fast extreme of the NMR timescale at 298.2 K (k_d≈ 10⁴ s^-1). These data are compared with those obtained in other solvents.     

Receptors for vasoactive intestinal peptide on isolated epithelial cells of rat ventral prostate

Receptors for porcine vasoactive intestinal peptide have been characterized in isolated epithelial cells of rat ventral prostate. The interaction of ¹²⁵I-labelled VIP with cells was rapid, reversible, specific, saturable and dependent on temperature. Degradation of peptide and receptors was minimized at 15°C. At apparent equilibrium, the binding of ¹²⁵I-labelled peptide was competitively inhibited by native VIP in the 1·10⁻¹⁰−10⁻⁷ M range concentration. The binding data were compatible with the existence of two classes of receptors: a high-affinity class with a K_d = 4.0 nM and a low binding capacity (0.12 pmol VIP/mg cell protein), and a low-affinity class with a K_d = 17.8 nM and a high binding capacity (1.6 pmol VIP/mg cell protein). Chicken VIP and porcine secretin exhibited a 7-fold higher and a 7-fold lower affinity than porcine VIP for binding sites, respectively. Glucagon, Leu-enkephalin, Met-enkephalin and somatostatin were ineffective. The presence of high-affinity receptors for VIP together with previous reports on the occurrence of VIP-containing neurones innervating the male genitourinary tract strongly suggest that this peptide may be important in the physiological regulation of the functions of prostatic epithelium.     

Synthesis and evaluation of phosphopeptides containing iminodiacetate groups as binding ligands of the Src SH2 domain

Phosphopeptide pTyr-Glu-Glu-Ile (pYEEI) has been introduced as an optimal Src SH2 domain ligand. Peptides, Ac-K(IDA)pYEEIEK(IDA) (1), Ac-KpYEEIEK (2), Ac-K(IDA)pYEEIEK (3), and Ac-KpYEEIEK(IDA) (4), containing 0–2 iminodiacetate (IDA) groups at the N- and C-terminal lysine residues were synthesized and evaluated as the Src SH2 domain binding ligands. Fluorescence polarization assays showed that peptide 1 had a higher binding affinity (K_d = 0.6 μM) to the Src SH2 domain when compared with Ac-pYEEI (K_d = 1.7 μM), an optimal Src SH2 domain ligand, and peptides 2–4 (K_d = 2.9–52.7 μM). The binding affinity of peptide 1 to the SH2 domain was reduced by more than 2-fold (K_d = 1.6 μM) upon addition of Ni²⁺ (300 μM), possibly due to modest structural effect of Ni²⁺ on the protein as shown by circular dichroism experimental results. The binding affinity of 1 was restored in the presence of EDTA (300 μM) (K_d = 0.79 μM). These studies suggest that peptides containing IDA groups may be used for designing novel SH2 domain binding ligands.     

DuP 753, the selective angiotensin II receptor blocker, is a competitive antagonist to human platelet thromboxane A2/prostaglandin H2 (TP) receptors

DuP 753 is a potent, selective angiotensin II type 1 (AT1) receptor antagonist. The possibility was investigated that DuP 753 may crossreact with thromboxane A₂/prostaglandin H₂ (TP) receptors. DuP 753 inhibited the specific binding of the TP receptor antagonist [³H]SQ 29,548 (5 nM) in human platelets with k_d/slope factor values of 9.6±1.4 μM/1.1±0.02. The AT2-selective angiotensin receptor ligand, PD 123,177 was a very weak inhibitor of specific [³H]SQ 29,548 binding in platelets (K_d/slope factor:200 μM/0.86). [³H]SQ 29,548 saturation binding in the absence and presence of DuP 753 resulted in an increase in equilibrium affinity constant (K_d: 9.3, 22, 33 nM, respectively) without a concentration-dependent reduction in binding site maxima (B_max: 3597, 4597, 3109 fmol/mg protein, respectively). Platelet aggregation induced by the TP receptor agonist U 46,619 was concentration-dependently inhibited by DuP 753 (IC₅₀=46 μM). These data indicate for the first time that DuP 753 is a weak but competitive antagonist at human platelet TP receptors.     

Insulin: Its binding to specific receptors and its stimulation of DNA synthesis and 2′,3′-cyclic nucleotide phosphohydrolase activity in cerebral cells cultured from embryonic mouse brain

The presence and specificity of insulin receptors was investigated in cultured cells obtained from 15–16 days old embryonic mouse cerebra. Developmental studies suggested that the maximum insulin binding occurred at about 11 days in vitro (DIV). Scatchard analysis of binding data revealed two types of binding sites. One type of receptor was the high affinity type (K _d=7.77×10^–9 M; number of receptor sites,B _max=350 fmol/mg protein) while the other type was of low affinity type (K _d=5.75×10^–8 M;B _max=1150 fmol/mg protein). The specificity of receptors for insulin was also confirmed by showing that [¹²⁵I]insulin was displaced by non-radioactive insulin but not by glucagon or growth hormone. Insulin displayed a clear dose-dependent stimulation of thymidine incorporation. It also stimulated the activity of the enzyme 2,3-cyclic nucleotide phosphohydrolase (CNPase), which is specifically associated with myelin produced from oligodendroglia. Thus insulin has a positive influence on the proliferation and differentiation of brain cells.     

Thyroid hormone: Aldosterone antagonism in cultured epithelial cells

Thyroid hormone (T₃) has been demonstrated to inhibit the action of aldosterone on sodium transport in toad urinary bladder and rat kidney. We have exammined the effect of T₃ on aldosterone action and specific nuclear binding in cultured epithelial cells derived from toad urinary bladder. In cell line TB6-C, addition of 5·10⁻⁸ M T₃ to culture media for up to 3 days results in no change in short-circuit current or transepithelial resistance. This concentration of T₃ completely inhibits the maximal increase in short-circuit current in response to 1·10⁻⁷ M aldosterone. The inhibition can be demonstrated with 18 h preincubation or with simultaneous addition of T₃ and aldosterone. The half-maximal concentration for the inhibition of the aldosterone effect is approx. 5·10⁻⁹ M T₃. T₃ has no effect on cyclic AMP-stimulated short-circuit current in these cells. The effect of T₃ on nuclear binding of [³H]aldosterone was examined using a filtration assay with data analysis by at least-squares curve-fitting program. Best fit was obtained with a model for two binding sites. The dissociation constants for the binding were K′_d1 = (0.82 ± 0.36)·10⁻¹⁰ M and K′_d2 = (3.2±0.60)·10⁻⁸ M.The half-maximal concentration for aldosterone-stimulated sodium transport in these cells is approx. 1·10⁻⁸ M. Analysis of nuclear aldosterone binding in cells preincubated for 18 h with 5·10⁻⁸ M T₃ showed a K′_d1 = (0.15 ± 0.10)·10⁻¹⁰ M and K′_d2 = (3.5 ± 0.10)·10⁻⁸ M. We conclude that T₃ i action of aldosterone on sodium transport at a site after receptor binding in the nucleus.     

Binding of a thromboxane A2/prostaglandin H2 agonist [H]U46619 to washed human platelets

The binding characteristics of [³H]U46619 to washed human platelets were studied. [³H]U46619 binding to washed human platelets was saturable and displaceable. Kinetic studies yielded a K_d of 11 ± 4 nM (n=4). Scatchard analysis of equilibrium binding studies revealed one class of high affinity binding sites with a K_d of 20 ± 7nM and a B_max of 9.1 ± 2.3 fmole/10⁷ platelets (550 ± 141 binding sites per platelet) (n=4). A number of compounds that act as either agonists or antagonists of the TXA₂/PGH₂ receptor were tested for their ability to inhibit the binding of [³H]U46619 to washed human platelets. The K_ds of the agonists and antagonists were similar to their potencies to induce or inhibit platelet aggregation. These data provide some evidence that [³H]U46619 binds to the putative human platelet TXA₂/PGH₂ receptor.     

Specific binding of [H]heparin to human carcinoma SW-13 and other mammalian cells

This study reports on the specific binding of [³H]heparin to human adrenocortical carcinoma cell line SW-13. Heparin binding to SW-13 cells is specific, saturable, and time- and temperature-dependent with maximum binding occurring between 90 and 120 min at 22 °C. Scatchard analysis revealed two classes of binding sites. The apparent K_d for high-affinity receptors is 2.14 × 10⁻⁸ M with 1.48 × 10⁶ sites per cells. Six other tested mammalian cell lines also have specific binding sites for heparin.     

Characterization of adrenergic receptors and related transduction pathways in the liver of the rainbow trout

Epinephrine (EPI) is thought to act by stimulating adenylyl cyclase (ACase) and cAMP production through β-adrenoceptors in the liver of more primitive vertebrates. Recent observations, however, point to an involvement of α₁-adrenoceptors in EPI action, at least in some fish species. The role of the α₁- and β-adrenergic transduction pathways has been investigated in rainbow trout (Oncorhynchus mykiss) hepatic tissue. Radioligand-binding assays with the β-adrenergic antagonist ³H-CGP-12177 using hepatic membranes purified on a discontinuous sucrose gradient confirmed the presence of β-adrenoceptors (K_d0.36 nM, B_max 8.61 fmol · mg⁻¹ protein). We provide the first demonstration of α₁-adrenoceptors in these same membranes; analysis of binding data with the α₁-adrenergic antagonist ³H-prazosin demonstrated a single class of binding sites with a K_dof 15.4 nM and a B_max of 75.2 fmol · mg⁻¹ protein. There is a straight correlation between β-adrenoceptor occupancy, ACase activation and cAMP production. On the contrary, the role of inositol 1,4,5-trisphosphate (IP₃) has to be elucidated; in fact, despite the presence of specific microsomal binding sites for IP₃ (K_d 6.03 nM, B_max 90.2 fmol · mg⁻¹ protein), its cytosolic concentration was not modulated by EPI. On the other hand, we have previously shown in American eel and bullhead hepatocytes that α₁-adrenergic agonists are able to increase intracellular concentrations of IP₃ and Ca²⁺ and to activate glycogenolysis. These data suggest a marked variation in the liver of different fish both in terms of α₁-binding sites affinity and of α₁-adrenoceptor/IP₃/Ca²⁺ transduction systems.     

Studies on (NA + K)-activated ATPase XLV. Magnesium induces two low-affinity non-phosphorylating nucleotide binding sites per molecule

ATP and adenylylimidodiphosphate (AdoPP[NH]P) bind to (Na⁺ + K⁺)-ATPase in the absence of Mg²⁺ (EDTA present) with a homogeneous but 15-fold different affinity, the K_d values being 0.13 μM and 1.9 μM, respectively. The binding capacities of the two nucleotides are nearly equal and amount to 3.9 and 4 nmol/mg protein or 1.7 and 1.8 mol/mol (Na⁺ + K⁺)-ATPase, respectively. The K_d value for ATP is equal to the K_m for phosphorylation by ATP (0.05–0.25 μM) and the binding capacity is equivalent to the phosphorylation capacity of 1.8 mol/mol (Na⁺ + K⁺)-ATPase. Hence, the enzyme contains two high-affinity nucleotide binding and phosphorylating sites per molecule, or one per α-subunit. Additional low-affinity nucleotide binding sites are elicited in the presence of Mg²⁺, as shown by binding studies with the non-phosphorylating (AdoPP[NH]P). The K_d and binding capacity for AdoPP[NH]P at these sites is dependent on the Mg²⁺ concentration. The K_d increases from 0.06 mM at 0.5 mM Mg²⁺ to a maximum of 0.26 mM at 2 mM Mg²⁺ and the binding capacity from 1.5 nmol/mg protein at 0.5 mM Mg²⁺ to 3.3 nmol/mg protein at 4 mM Mg²⁺. Extrapolation of a double reciprocal plot of binding capacity vs. total Mg²⁺ concentration yields a maximal binding capacity at infinite Mg²⁺ concentration of 3.8 nmol/mg protein or 1.7 mol/mol (Na⁺ + K⁺)-ATPase. The K_d for Mg²⁺ at the sites, where it exerts this effect, is 0.8 mM. The K_d for the high-affinity sites increases from 1.5–1.9 μM in the absence of Mg²⁺ to a maximum of 4.2 μM at 2 mM Mg²⁺ concentration. The binding capacity of these sites (1.8 mol/mol enzyme) is independent of the Mg²⁺ concentration. Hence, Mg²⁺ induces two low-affinity non-phosphorylating nucleotide binding sites per molecule (Na⁺ + K⁺)-ATPase in addition to the two high-affinity, phosphorylating nucleotide binding sites.     

Volume regulation of human peripheral blood lymphocytes and stimulated proliferation of volume-adapted cells

Human peripheral blood lymphocytes exposed to hypotonic media (Ca/Mg-free, room temp.) first swell and then shrink. This shrinking response is characterized by a simple exponential with a half-time of 1.44±0.60 min (n=11) and its extent but not the half-time for a given hypotonicity is influenced by [K⁺]₀. Using K-selective electrodes, we observe a change in [K⁺]₀ when cells are diluted into hypotonic media. A half-time of 1.55±0.06 min (n=4) was obtained. A similar half-time was obtained by assay of total cell K using atomic absorption spectroscopy. At all osmolarities [K⁺]_i was decreased from control values and was constant as [K⁺]₀ was increased. Short-term incubation with ouabain (10⁻⁴ M) had no effect. Decreasing osmolarities progressively inhibited phytohemagglutinin-stimulated DNA synthesis, yet cell number and viability remained unaltered. Our evidence indicates that the volume response is mediated by a change in the passive permeability of the plasma membrane to K and/or to the accompanying anions, and that the consequently volume-adapted cells are growth-inhibited.     

Characterization of [3H]LS‐3‐134, a novel arylamide phenylpiperazine D3 dopamine receptor selective radioligand

LS‐3‐134 is a substituted N‐phenylpiperazine derivative that has been reported to exhibit: (i) high‐affinity binding (K_i value 0.2 nM) at human D3 dopamine receptors, (ii) > 100‐fold D3 versus D2 dopamine receptor subtype binding selectivity, and (iii) low‐affinity binding (K_i > 5000 nM) at sigma 1 and sigma 2 receptors. Based upon a forskolin‐dependent activation of the adenylyl cyclase inhibition assay, LS‐3‐134 is a weak partial agonist at both D2 and D3 dopamine receptor subtypes (29% and 35% of full agonist activity, respectively). In this study, [³H]‐labeled LS‐3‐134 was prepared and evaluated to further characterize its use as a D3 dopamine receptor selective radioligand. Kinetic and equilibrium radioligand binding studies were performed. This radioligand rapidly reaches equilibrium (10–15 min at 37°C) and binds with high affinity to both human (K_d = 0.06 ± 0.01 nM) and rat (K_d = 0.2 ± 0.02 nM) D3 receptors expressed in HEK293 cells. Direct and competitive radioligand binding studies using rat caudate and nucleus accumbens tissue indicate that [³H]LS‐3‐134 selectively binds a homogeneous population of binding sites with a dopamine D3 receptor pharmacological profile. Based upon these studies, we propose that [³H]LS‐3‐134 represents a novel D3 dopamine receptor selective radioligand that can be used for studying the expression and regulation of the D3 dopamine receptor subtype.     

A dispersed-whole cell method for the determination of androgen receptors in human skin fibroblasts

A method is described for the determination of binding capacity (R_o) and dissociation constant (K_d) of androgen receptors in dispersed, whole, cultured human skin fibroblasts. The cells, obtained from punch biopsies or operative specimens of skin, are grown to confluence in monolayers, harvested, washed, and dispersed in medium. Binding is assessed by incubating the cells with [³H]dihydrotestosterone³ (DHT) at 22°C for one hour followed by washing, centrifugation and counting. Non-specific binding is determined by adding radioinert methyltrienolone (R1881). Scatchard plots are constructed using 6 concentration points; binding is expressed as sites/cell. The method is precise (R_o = 12,105 ± 4305 (S.D.) sites/cell; K_d = 1.0 ± 0.7 (S.D.) × 10^?9M, n = 14 for one prepuce cell line) and independent of cell passage number. Binding is linear with respect to cell number and shows those characteristics commonly attributed to androgen receptors: high affinity, low binding capacity, saturable nuclear binding, androgen specificity, and an 8S peak in fibroblast cytosol using sucrose density gradients. Cell lines shown by other published methods to lack androgen receptors had no detectable DHT binding with this method. This assay technique has the advantages of simplicity, rapidity, and precision.     

A new nonhydrolyzable reactive cGMP analogue, (Rp)-guanosine-3',5'-cyclic-S-(4-bromo-2,3-dioxobutyl)monophosphorothioate, which targets the cGMP binding site of human platelet PDE3A

The amino acids involved in substrate (cAMP) binding to human platelet cGMP-inhibited cAMP phosphodiesterase (PDE3A) are identified. Less is known about the inhibitor (cGMP) binding site. We have now synthesized a nonhydrolyzable reactive cGMP analog, Rp-guanosine-3′,5′-cyclic-S-(4-bromo-2, 3-dioxobutyl)monophosphorothioate (Rp-cGMPS-BDB). Rp-cGMPS-BDB irreversibly inactivates PDE3A (K_I = 43.4 ± 7.2 μM and k_cart = 0.007 ± 0.0006 min⁻¹). The effectiveness of protectants in decreasing the rate of inactivation by Rp-cGMPS-BDB is: Rp-cGMPS (K_d = 72 μM) > Sp-cGMPS (124), Sp-cAMPS (182) > GMP (1517), Rp-cAMPS (3762), AMP (4370 μM). NAD⁺, neither a substrate nor an inhibitor of PDE3A, does not protect. Nonhydrolyzable cGMP analogs exhibit greater affinity than the cAMP analogs. These results indicate that Rp-cGMPS-BDB targets favorably the cGMP binding site consistent with a docking model of PDE3A-Rp-cGMPS-BDB active site. We conclude that Rp-cGMPS-BDB is an effective active site-directed affinity label for PDE3A with potential for other cGMP-dependent enzymes.     

Dopamine-stimulated cyclic AMP and binding of [H]dopamine in acini from dog pancreas

Dispersed acini from dog pancreas were used to examine the ability of dopamine to increase cyclic AMP cellular content and the binding of [³H]dopamine. Cyclic AMP accumulation caused by dopamine was detected at 1·10⁻⁸ M and was half-maximal at 7.9±3.4·10⁻⁷M. The increase at 1·10⁻⁵ M, (7.5-fold) was equal to the half-maximal increase caused by secretin at 1·10⁻⁹ M. Haloperidol, a dopaminergic receptor antagonist inhibited cyclic AMP accumulation caused by dopamine. The IC₅₀ value for haloperidol, calculated from the inhibition of cyclic AMP increase caused by 1·10⁻⁵ M dopamine was 2.3±0.9·10⁻⁶M. Haloperidol did not alter basal or secretin-stimulated cyclic AMP content. [³H]Dopamine binding was studied on the same batch of cells as cyclic AMP accumulation. At 37°C, it was rapid, reversible, saturable and stereospecific. The K_d value for high affinity binding sites was 0.43±0.1·10⁻⁷M and 4.7±1.6·10⁻⁷M for low affinity binding sites. The concentration of drugs necessary to inhibit specific binding of dopamine by 50% was 1.2±0.4·10^/t-7M noradrenaline, 2·10^/t-7 M epinine, 4.1±1.8·10^/t-6M fluphenazine, 8.0±1.6·10^/t-6M haloperidol, 4.2±1.2·10⁻⁶Mcis-flupenthixol, 2.7±0.4·10⁻⁵Mtrans-flupenthixol, >1·10⁻⁵M apomorphine, sulpiride, naloxone and isoproterenol.